Field of the Invention
The current invention relates to compositions of matter that are able to kill (or inhibit) microbes, and have low mammalian toxicity. The current invention also relates to certain compositions and their uses in a variety of settings including but not limited to preservatives, antiseptics, and the prevention and treatment of wound infections, as well as other infectious diseases.
Discussion of Related Art
Cationic Antimicrobials have Demonstrated Utility; Toxicity is a Problem.
For over half a century, cationic (positively charged) antimicrobials have been used in a variety of medical and non-medical settings, ranging from systemic antibiotics to industrial cleansers. Cationic antimicrobials bind preferentially to bacterial membranes, which typically display more negative charge than mammalian membranes. This interaction can disrupt membrane function and potentially lead to bacterial cell death. Cationic antimicrobial compounds include certain antibiotics (e.g., polymyxins), bisbiguanides (e.g., chlorhexidine), polymeric biguanides (e.g., polyhexamethylene biguanide), and quaternary ammonium compounds (QAC) (e.g., benzalkonium chloride), as well as natural antimicrobial peptides (AMPs) (e.g., defensins). While each class of cationic antimicrobial compounds has demonstrated antimicrobial activity in one or more settings, toxicity has been a consistent problem.[1-12]
Polymyxins, produced by Bacillus polymyxa, are cyclic peptides with hydrophobic tails.[6, 7] The cyclic peptide portion (approx. 10 amino acid residues; positively charged) interacts strongly with negatively charged lipopolysaccharide (LPS) found on the outer membrane of Gram-negative bacteria. The hydrophobic tail is thought to interact with, and in some cases, disrupt the bacterial membrane. Polymyxins have antimicrobial activity against many Gram-negative bacteria, including Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Enterobacter species, but have limited activity against Proteus, most Serratia, or Gram-positive bacteria [7]. Significant neurotoxicity and nephrotoxicity have contributed to their limited use as systemic antibiotics [13]. Today, Polymyxins are sometimes used as a last resort for Gram-negative infections that are highly antibiotic resistant, such as those caused by multi-drug resistant P. aeruginosa. They are also used as topical antimicrobial agents for small cuts and scrapes of the skin.
Chlorhexidine is widely used in the pre-operative surgical setting as an antiseptic cleanser for general skin cleaning, preoperative bathing, and surgical site preparation [7]. Chlorhexidine is active against a wide range of Gram-positive and Gram-negative bacteria, although resistance by some Gram-negative bacteria (e.g., P. aeruginosa, Providentia species) has been reported [5, 10]. Formulations containing 2-4% chlorhexidine appear to be most effective as antimicrobials, but can cause skin irritation. Overall, chlorhexidine is relatively safe when applied to intact skin because minimal amounts of the compound are absorbed. However, due to irritation and toxicity, chlorhexidine is contraindicated for use near the eyes, ears, brain tissues, and meninges [2]. Low concentrations (e.g., 0.05% to 0.12%) are sometimes used as wound washes and oral rinses. Activity is pH dependent, as low pH environments reduce activity. In addition, chlorhexidine is not compatible with anionic compounds (e.g., hard water, soap, alginate) and shows reduced activity in the presence of organic materials (e.g., blood).
Polyhexamethylene biguanide (PHMB) has been used in diverse consumer applications for over 40 years. PHMB is used in swimming pool sanitizers, preservatives of plasticized PVC, and general-purpose environmental biocides [1]. Early production of PHMB resulted in highly polydisperse oligomers with molecular weights ranging from 500-6,000 g/mol. Limited chemical characterization largely precluded early PHMB use in pharmaceutical products. Recent PHMB formulations have been able to address polydispersity. Similar to chlorhexidine, use of PHMB is contraindicated for eyes, ears, brain tissues, meninges, and joints [4].
Quaternary ammonium compounds (QACs) are amphoteric surfactants, typically containing one nitrogen atom linked directly to four alkyl groups, which may vary in hydrophobic structure [1, 2]. QACs are primarily bacteriostatic, but at higher concentrations can be bacteriocidal against certain organisms. QACs are antimicrobial against Gram-positive bacteria, but are less effective against Gram-negative bacteria (e.g., P. aeruginosa). Because of weak activity against Gram-negatives, QACs are generally not used in health-care settings for hand antisepsis. Several outbreaks of infection have been traced to QAC compounds contaminated with Gram-negative bacilli [8]. QACs appear to be more susceptible to resistance mechanisms mediated through multidrug efflux pumps. Activity is also greatly reduced in the presence of organic matter.
Natural antimicrobial peptides (AMPs) are often cationic. Natural antimicrobial peptides (AMPs) (typically, less than 50 amino acids) are widely distributed in most species from insects to mammals, and are thought to play key roles in innate immunity [14]. AMPs have demonstrated potent killing/inhibition of bacteria, viruses, fungi and parasites [15]. AMPs are thought to be important in preventing and controlling infections. AMPs are heavily deposited at interfaces such as the skin, respiratory tract, and gastrointestinal lining, and are released by white blood cells at sites of inflammation. White blood cells use AMPs as part of their direct killing mechanisms in phagolysosomes. Certain AMPs contribute to the regulation of inflammation and adaptive immunity [15]. In addition, AMPs have demonstrated inhibitory activity against spermatozoa and cancer cells.
Most AMPs share structural characteristics leading to physical, receptor-independent modes of killing [9]. A widely accepted mechanism of action of AMPs is microbial membrane disruption or perturbation (followed sometimes by pore formation) leading to cell death. Typically, AMPs contain positively charged and hydrophobic domains that are spatially segregated—cationic amphiphiles. Substantial hydrophobic content of AMPs (typically, 30 to 60% mole fraction) is an important feature for antimicrobial activity as it “governs the extent to which a peptide can partition into the lipid bilayer” [16]. AMPs that form alpha-helices “frequently exist as extended or unstructured conformers in solution” and become helical “upon interaction with amphipathic phospholipid membranes” [16]. This suggests that the “local environment at the bacterial outer surface and membranes is important and can induce antimicrobial peptide conformational changes that are necessary for peptide attachment to and insertion into the membrane” [3].
Nisin (a bacterially-derived AMP that has been used as a food preservative) was shown to be a weak emulsifying agent for oil-water mixtures, the process being significantly pH- and temperature-dependent [17].
Several natural AMPs and related technologies have been patented. Lehrer and Selsted disclosed AMP sequences analogous to those of defensins isolated from macrophages (U.S. Pat. No. 4,543,252). The magainin class of AMPs, first isolated from the skin of certain frogs, has been described by Zasloff (U.S. Pat. No. 4,810,777). Modified magainins, particularly sequence deletions or substitutions, have also been described (e.g., U.S. Pat. Nos. 4,962,277; 5,221,732; 5,912,231; and 5,792,831). Selsted and Cullor disclosed bovine indolicidin AMP as a broad-spectrum antimicrobial compound (U.S. Pat. No. 5,324,716).
Synthetic Peptide-Based Cationic Oligomers May Function as Antimicrobials.
Salick and colleagues have disclosed a sequence-specific beta-hairpin peptide (20-mer) which can form an antimicrobial hydrogel in the presence of sufficient salt concentration (US Published Patent Application No. 2011/0171304). When the peptide is “dissolved in water, it remains unfolded and soluble due to the charge repulsion between positively charged side chains.” The addition of salt is thought to “screen the side chain-derived charge and allow the peptide to fold” into a beta-hairpin which may “assemble into a network of beta-sheet rich fibrils.” The peptide consists of 60% hydrophobic content and contains two arginine residues that seem to be important for effective antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). The peptides themselves do not appear to be inherently antimicrobial, as the inventors have reported that “peptide diffusing from the gel is not the active agent.” When S. aureus was subjected to 100 μM (approx. 230 μg/ml) aqueous solutions (i.e., not hydrogels) of peptide, “bacterial proliferation was minimally affected.” Thus, for antimicrobial activity, bacteria must directly contact the hydrogel surface; “folded but not gelled” peptide does not inhibit bacterial proliferation. Similar findings were reported for other closely-related beta-hairpin peptides [18].
Gellman and coworkers have disclosed antimicrobial compositions containing beta-amino acid oligomers (U.S. Pat. Nos. 6,060,585; 6,683,154; US Published Patent Application Nos. 2007/0087404; 2008/0166388) with well-defined secondary structures. The beta-peptides contain ring structures in the peptide backbone which limit conformational flexibility. DeGrado and coworkers have also described antibacterial beta-peptides, containing oligomers (7-mer or shorter) of a tri-beta-peptide (U.S. Pat. No. 6,677,431).
Other synthetic peptide-based compounds that may mimic overall structure of natural AMPs have been described. DeGrado reported amphiphilic sequence-random beta-peptides based on structural properties of the natural AMPs magainin and cecropin [19]. Gellman and coworkers have described a random-sequence, beta-peptide oligomer with an average length of 21 residues, polydispersity index (Mn/Mw) of 1.4, and 40% hydrophobic residues [20]. In other studies, Gellman identified helical beta-peptides [19]. A 60% “hydrophobic face” along the helical cylinder was found to have optimal antimicrobial activity, while a 40% face displayed low activity.
Synthetic Cationic Polymers Comprised of Non-Natural Building Blocks May Function as Antimicrobials.
Several classes of synthetic antimicrobial polymers with non-natural building blocks or repeat-units have been described; they are the subject of a 2007 review by Tew [22]. These polymers are comprised of structures/monomeric units that are not found in nature. These non-natural polymers often feature easy and cost-efficient syntheses, and stability against enzymatic degradation. However, limitations of these and other non-natural polymers may include limited antimicrobial activity, as well as a lack of biocompatibility and biodegradability. Materials in this class are comprised of unnatural building blocks (e.g. aryl amides, highly conjugated aromatic groups) and are considered outside the scope of this invention [21-25]. (For examples, see U.S. Pat. No. 7,173,102; US Published Patent Application Nos. 2008/0176807; 2010/0105703).
Antimicrobial peptoids (N-substituted glycines) have been described by Winter and coworkers [28]. A series of short (3-monomer) peptoids were tested against a broad spectrum of Gram-positive and Gram-negative bacteria, and hemolytic activity (HC50) was lower than antimicrobial activity (minimum inhibitory concentrations, MICs). A representative tri-peptoid protected S. aureus-infected mice in vivo in a simple infection model.
Synthetic Methodologies for Copolypeptides (Deming Method).
Traditional synthetic methodologies have precluded the efficient synthesis of oligopeptide libraries with orthogonal (or semi-orthogonal) modification of multiple properties. Important properties to be modified include amino acid sequence, overall chain length, and ratio of cationic to hydrophobic amino acids. Moreover, the practical, cost-effective synthesis of low polydispersity (PDI between 1.0 and 1.4) copolypeptide mixtures has also not been easily accessible [25].
Control over multiple properties, and the ability to create low polydispersity compounds, would allow optimization of multiple structure-function relationships. A major challenge in synthetic polypeptide AMP research is prohibitive production costs in solid-phase synthesis. In addition, significant chemical limitations of both solid-phase and solution-phase synthetic methods include lack of control over chain growth. This leads to chain branching, polydispersity and low product yields.
In 1997, Deming developed well-defined initiators to polymerize amino acid derivatives into oligopeptide chains [25, 26]. This methodology added amino acid monomers to a growing chain in batches. The initiators were transition-metal complexes that allowed controlled synthesis to yield high molecular weight, narrowly-distributed, multi-block polypeptide formulations. The initiators and synthetic methods are well described in the literature and in several patents (U.S. Pat. Nos. 6,680,365; 6,632,922; 6,686,446; 6,818,732; 7,329,727; US Published Patent Application No. 2008/0125581).
Typically, the synthetic polypeptides have a simple binary composition (e.g., lysine (K), leucine (L) copolymers). Amphiphilic polypeptides contain ionic amino acid monomers (e.g., lysine, arginine (R), glutamate (E)) co-polymerized with neutral hydrophobic amino acids (e.g., leucine, alanine (A)). By variation of method of monomer addition, copolymerizations may be conducted to obtain sequences of amino acid residues along the copolymer chain that are blocky, random, or a combination of both (i.e. blocks of random sequences).
Random Synthetic Copolypeptides in Solution Demonstrate Antimicrobial Activity.
The Deming laboratory has observed antimicrobial activity for a series of water-soluble copolypeptides containing varying ratios of cationic (lysine, (K)) and hydrophobic (leucine (L), isoleucine (I), valine (V), phenylalanine (F), or alanine (A)) amino acids that were randomly arranged [27]. Copolypeptides demonstrated varying antimicrobial activity against S. aureus (Gram-positive), P. aeruginosa (Gram-negative), and E. coli (Gram-negative) in suspension growth assays. Lysine-alanine copolypeptides demonstrated a broad “toxic effect on all three species of bacteria studied” and were concluded to be the “most effective antimicrobial copolymer combination.” Circular dichroism spectra of lysine-alanine and lysine-leucine copolypeptides showed “unambiguous random coil conformations when free in solution.” This work did not examine the antimicrobial activity of synthetic block sequence copolypeptides or synthetic copolypeptides deliberately formulated as micelles, or incorporated into emulsions/nanoemulsions (also see [28, 29]).
Using Deming synthesis methods, Chan-Park and colleagues recently studied the antimicrobial activity of soluble, random-sequence copolypeptides containing 2-3 different amino acids [26]. Random 25-mer copolypeptides, comprised of lysine-phenylalanine or lysine-phenylalanine-leucine, demonstrated the broadest activity against five microbes and had the lowest MICs. The effects of total peptide length and hydrophobic content on antimicrobial activity were investigated. Lysine-phenylalanine copolypeptide was reported to have “broader antibacterial activity when it is 25 residues long than at shorter or longer length.” Optimum hydrophobic content for lysine-phenylalanine compounds (and other random copolypeptides) was found to be about 60%. However, optimized lysine-phenylalanine and lysine-phenylalanine-leucine compounds showed high hemolytic activity compared to other natural and synthetic peptides. The authors suggested that the compounds' “high hydrophobicity (60%) or more hydrophobic species present may have resulted in high toxicity to mammalian red blood cells.” In addition, lysine-alanine and lysine-leucine random copolypeptides showed no significant activity against the fungal organism Candida albicans. Circular dichroism analysis indicated that lysine-phenylalanine and lysine-phenylalanine-leucine random copolypeptides show “lack of a distinct secondary structure” and do not form alpha-helices or beta-sheets.
Synthetic Copolypeptides can be Formulated to Achieve Hierarchical Structures.
The presence of both polyelectrolyte and hydrophobic domains leads to microphase segregated materials. Resulting superstructures can include multimers in solution, micelles, emulsions (with oil), sheets, vesicles and fibrils that form hydrogels. Self-assembly into different hierarchical structures can be controlled by: varying composition and chain length; varying concentration; presence of L-, D-, or racemic amino acids; and modification of side-chains and chain-termini (e.g. polyethylene glycol (PEG)). Secondary structure of hydrophobic domains (i.e. random coil vs. alpha-helix) plays an important role in superstructure formation. The nature of the hydrophobic domain or polymer segments determines the type of intermolecular interactions that are established between chains. These attractive interactions are balanced by the interactions with the solvent. There exists an equilibrium between the free energy of self-association with the free energy of hydration for each molecule and for each fragment of the supermolecule.
Synthetic copolypeptides can also be designed to form hydrogels. Certain characteristics, such as long-hydrophilic blocks (cationic or anionic) and ordered hydrophobic blocks (e.g., alpha-helical) were shown to favor hydrogel formation. Studies suggest that several synthetic copolypeptide-based hydrogels, including K180L20 (and other KxLy) block copolypeptides, are biocompatible in vivo. Deming et al. previously reported that block copolypeptide hydrogels can serve as tissue scaffolds in the murine central nervous system (CNS) [27]. Hydrogels were injected into mouse forebrain and created 3D gel deposits in vivo. Toxicity, inflammation and gliosis were minimal and similar to saline controls. After 8 weeks, in many cases, copolypeptide deposits were vascularized with cell density similar to adjacent tissue, suggesting hydrogels are supportive of cellular migration and proliferation.
Deming (PCT publication WO 2009/025802) disclosed nanoemulsions and double nanoemulsions stabilized by synthetic block copolypeptides [27]. Antimicrobial activity of the emulsified copolypeptides was not disclosed therein.
Nanoemulsions prepared without copolypeptides can display some antimicrobial activity. Baker and coworkers have focused on the use of nanoemulsions as antimicrobial agents. They reported antimicrobial emulsions stabilized by phosphate-based or other small molecule surfactants (U.S. Pat. Nos. 6,015,832; 6,506,803; 6,559,189; 6,635,676; 5,618,840; 5,547,677; and 5,549,901).
Potential relationships between antimicrobial activity and/or mammalian cell toxicity of cationic amphiphiles and their assembly into higher-order structures are not well understood. Limited relevant information has been reported. For example, the antimicrobial activity of epsilon-poly-lysine (EPL) was slightly reduced by coordination to a lipid and emulsification, relative to free EPL in solution [33].